MicroSolv Technologies Knowledge Base

Microsolv

Browse Ask a Question
 
Tools
Categories

Fast Analysis of Human Normal Immunoglobulin using CElixirPro3

Reference Number: AA-01408 Created: 04/30/2013 11:24 AM Last Updated: 04/30/2013 12:06 PM

Introduction
Human normal immunoglobulin for intravenous administration (IVIg) is a liquid or freeze-dried preparation containing mainly immunoglobulin G (IgG), while albumin may be added as stabilizer (Ph. Eur.). Monographs describe that the protein composition should be examined by zone electrophoresis. Traditionally this is done using cellulose acetate gel or agarose gel and barbital buffer. The method is the same as for serum protein analysis (Figure 1) and quantitation can be done using a gel scanner (Figure 2). We describe here a method using capillary electrophoresis and CElixirPro3™ buffer kit.

Figure 1. Agarose gel electrophoresis of serum proteins.

Figure 2. Gel scan of serum proteins.

Methods and Separation
Instrumentation

Suitable instruments are capillary electrophoresis such as PA800 plus or P/ACE MDQ (Beckman Coulter Brea CA, USA) using a UV detector at 200 nm. The capillary is fused silica (Polymicro Phoenix AZ, USA) 50μm internal diameter, 60.2 cm total length and 10 cm to the detector as the short end injection side is used.
Method
The buffer used is CElixirPro3™ buffer at pH 6.2 (obtained by mixing 2 g of pH 4 buffer with 8 g of pH 7.2 buffer from the kit). The samples are diluted (40μL—360 μL) with the sample diluent from the kit. Before separation the capillary is coated with CElixirPro3™ Indicator solution and CElixirPro3™ Buffer as described in the instructions for use. Sample is injected at the short end for 8 sec at 0.5 psi, followed by a water plug (7 sec at 0.2 psi) Separation is done at 12 or 15 kV. After separation the capillary is rinsed with 0.1 M NaOH.
Separation of BRP
Figure 3 shows an overlay of 4 separations of the Human Immunoglobulin for  electrophoresis BRP batch 2 reference standard. The first peak represents the immunoglobulin peak and the second the albumin peak.

Figure 3. Analysis of Human immunoglobulin BRP reference solution (EDQM Reference standard). Overlay of 4 runs.


Quantification of albumin in IVIg
Figure 4 shows the separation of a sample containing 100 mg/mL of IgG and an increasing quantity of albumin between 0.5 mg/mL and 10 mg/mL. The calibration curve is shown in Figure 5.


Figure 4. Overlay of blood immunoglobulin with increased amount of albumin.

Figure 5. Calibration of albumin between 0.5 and 10 mg/mL showing a goodness of fit R2 > 0.99.

Summary
The CElixirPro3™ kit allows easy and fast analysis by capillary electrophoresis of different human immunoglobulin (IVIg) for quantitation.


     © Copyright MicroSolv Technology Corporation, all rights reserved.

Info Ask a Question 
Your Email: Subject: Question: